In U.S. Pat. No. 5,010,063 a description was given of a structural modification, in basic medium, of glycosaminoglycans with heparin and heparan structure with subsequent isolation from the reaction mixture of new derivatives with respect to the state of the art, as demonstrated unmistakably by the chemical and physical characteristics and especially by the .sup.13 C-NMR spectrum.
In the subsequent U.S. Pat. No. 5,104,860 a further structural modification was described, in a basic or neutral medium, which, starting from the products formed in the reaction conditions described in U.S. Pat. No. 5,010,063, and from the glycosaminoglycans with heparin or heparan structure used as starting products in the same patent, originated a range of new products, different from those described in said patent and new with respect to the state of the art, as demonstrated unmistakably by the chemical and physical characteristics and especially by the .sup.13 C-NMR spectrum.
The chemical and physical characteristics of the products described in U.S. Pat. No. 5,010,063 and the results of a subsequent structural study described by Jaseia M., Rej R., Sauriol F., Perlin A. S. in Can. J. Chem 67, 1449-56 (1989), with the specific aim of explaining the mechanism of the reaction of structural modification in a basic medium, have demonstrated that these derivatives show a modification which concerns just one of the saccharide units characteristic of glycosaminoglycans with heparin or heparan structure, more specifically the unit of .alpha.-L-iduronic acid sulfated in position 2 and involving its transformation into a 2,3-epoxygulonic unit. The so obtained epoxides are represented by the following general formula IV ##STR3##
Likewise it has been demonstrated that semi-synthetic glycosaminoglycans with one 2,3-epoxygulonic unit and also glycosaminoglycans with heparin or heparan structure, in conditions of reaction similar to those described in U.S. Pat. No. 5,104,860 undergo a structural modification which also concerns the saccharide unit of .alpha.-L-iduronic acid sulfated in position 2 and involving the transformation of this saccharide unit into a unit of non-sulfated .alpha.-L-iduronic acid or .alpha.-L-galacturonic acid, according to the conditions of reaction used.
So U.S. Pat. No. 5,010,063 describes semi-synthetic glycosaminoglycans containing an epoxy function between positions 2 and 3 of the unit of .alpha.-L-iduronic-2-O-sulfate acid taken as a starting point and the conditions of reaction necessary for obtaining them, while U.S. Pat. No. 5,104,860 describes products deriving from further transformation of the epoxide, confirmed as having one unit of non-sulfated .alpha.-L-iduronic or .alpha.-L-galacturonic acid, and the conditions of reaction necessary for obtaining them starting from the epoxide itself or, as an alternative, starting from the glycosaminoglycans with heparin or heparan structure themselves, used as starting products in U.S. Pat. No. 5,010,063.
Subsequently, in published European patent application EP 565.862, semi-synthetic glycosaminoglycans were described in which one of the saccharide units characteristic of the glycosaminoglycans with heparin or heparan structure, more specifically that containing .alpha.-L-iduronic-2-O-sulfate acid, has undergone, entirely or in part, a structural modification with transformation into .alpha.-L-galacturonic acid substituted with a nucleophilic radical in position 3. The process claimed in said published European patent application describes the obtaining of the semi-synthetic glycosaminoglycans of general formula III ##STR4## by treating the epoxides of formula IV, described in U.S. Pat. No. 5,010,063, with a nucleophilic reagent.
Object of the present invention is a new process for the preparation of the semi-synthetic glycosaminoglycans of general formula III directly starting from the glycosaminoglycans with heparin or heparan structure of general formula I ##STR5##
The configuration of the uronic residue different from that of the glycosaminoglycans with heparin or heparan structure was determined on the basis of the chemical physical data, particularly on the basis of the .sup.13 C-NMR spectrum.
This new process represents an improvement of the process described in the published European patent application EP 565.862 because it uses as starting product the glycosaminoglycan of formula I, while in said European patent application the starting material was the epoxy derivative of formula IV in its turn obtained by the glycosaminoglycan of formula I according to the process described in U.S. Pat. No. 5,010,063. The advantage of directly obtaining the product of formula III in only one reaction by starting from the glycosaminoglycan of formula I instead of obtaining it by means of two consecutive reactions, the first of which includes the process of synthesis, isolation and purification of the epoxide of formula IV starting from the glycosaminoglycan of formula I, is evident in terms of overall yield and of industrial cost.
To better define the field of the present invention, we would like to point out that the expression glycosaminoglycans with heparin or heparan structure is intended to indicate polysaccharides with a molecular weight of between about 3000 and about 50000 Daltons and characterized by the fact of possessing a disaccharide unit consisting of a uronic acid (which may be .alpha.-L-iduronic or .beta.-D-glucuronic) and of .alpha.-D-glucosamine, connected, in alternate sequences, by 1,4-glycosidic bonds as described by Gallagher J. T. and Walker A. in Biochem. J., 230, 665-674, (1985), Lindhal U., Kjellen L. in Thrombosis and Haemostasis 66, 44-48 (1991) and by Turnbull J. E., Gallagher J. T. in Biochem. J. 273, 553-559 (1991). Since the .alpha.-L-iduronic acid can be sulfated in position 2 and the glucosamine can be N-acetylated, N-sulfated, 6-O-sulfated, 3-O-sulfated, according to the variable positions of the substituents, at least 10 different disaccharide units are possible, whose combination may generate a large number of different sequences. Bearing in mind the most represented disaccharide units and the most frequent sequences, we can say with reasonable approximation, that the general formula I can be attributed to glycosaminoglycans with heparin or heparan structure ##STR6## where R represents hydrogen or the sulfate residue (SO.sub.3.sup.-) and where m and n are whole numbers between 1 and 100.
In heparin structured glycosaminoglycans of natural origin the value of m is high and the disaccharide unit A represents about 80% of the disaccharide units: on the contrary, in heparan structured glycosaminoglycans of natural origin the value of n is high and the disaccharide unit B represents about 80% of the disaccharide units.
The general formulae I and III are intended to reveal the composition of the main saccharide units but make no reference to their sequence. As is known to experts in the art, it is possible to make a chemical modification of glycosaminoglycans of natural origin, for example through reactions of N-desulfatation, possibly followed by reactions of N-acetylation, thus also obtaining semi-synthetic N-desulfated heparins or N-desulfated-N-acetylated heparins. In addition, these glycosaminoglycans, whether natural or semi-synthetic, may be subjected to depolymerization processes by means of which the molecular weight is taken to levels generally between 3000 and 10000 Daltons.
The structural modification described in this invention for obtaining new semi-synthetic glycosaminoglycans involves the partial or total transformation of the saccharide unit of .alpha.-L-iduronic-2-O-sulfate acid into a saccharide unit of .alpha.-L-galacturonic acid substituted by a nucleophilic radical in position 3, with the subsequent disappearance of the heparin or heparan structure. This structural modification can be done on any type of compound with heparin or heparan structure. Indeed, besides being selective, the chemical process described in this invention can be applied to glycosaminoglycans with heparin or heparan structure which present all the possible sequences; i.e. it is independent of the type and of the level of functionalization of the saccharide unit which precedes or follows in the sequence the unit of .alpha.-L-iduronic-2-O-sulfate acid which is the object of the reaction of structural modification.
The structure of the new products is represented by the general formula III ##STR7## where p+q=m, with p other than 0, and m, n and R have the meaning as seen above, and where --Z(R.sub.2)R.sub.1 represents the nucleophilic group introduced through the process described in this invention.
The reaction of structural modification which involves the modification from saccharide unit of .alpha.-L-iduronic-2-O-sulfate acid into saccharide unit of .alpha.-L-galacturonic acid, with the introduction of the nucleophilic radical in position 3 of the .alpha.-L-galacturonic acid, does not lead to the depolymerization of the glycosaminoglycans or alteration in the distribution of the molecular weight of the polysaccharide chains which form them, and for this reason the present reaction can be applied to glycosaminoglycans with heparin or heparan structure of any molecular weight. The products obtained can however be subjected to the known processes of chemical or enzymatic depolymerization.